I’m having some problems with obtaining integrants for a few strains and was hoping someone might have some suggestions or ideas.
I have previously performed a few dozen integrations with success. I haven’t changed my protocol and suddenly have been running into trouble getting
integrated lines. I am, however, getting extremely high transmitting (>>99%) lines that are very bright and could easily be mistaken for integrants without extreme
scrutiny.
I appreciate any suggestions or ideas as to why this might be happening. Thank you.
What co-injection marker are you using? I did some integrations with rescue of lin-15AB(n765ts) awhile back and along with real integrants I got some lines that behaved as you describe, that I took to be synthetically lethal with n765. But if you’re just using the GFP marker, or using something less prone to synthetic lethals than lin-15B, this is less likely the case.
I’m using mCherry as a co-injection marker and worms injected are WT. Worms with the array prior to mutagenesis are healthy.
Is it possible that this could be caused by an inadequate quantity of UV joules delivered? I am successfully enriching for a high transmission rate, but does this necessarily
mean that an adequate amount of energy was delivered (I use a UV Stratalinker with energy setting on 300).
The array could be toxic when integrated because expression becomes more uniform. As an array the toxicity is mitigated because of mosaic expression/inheritance. I have seen this with sur-5- or eft-3-driven markers: Your integrants are slower-growing so you are less likely to single them after ionizing radiation treatment, hence you get only “almost-integrants” out of such a screen. You could repeat the screen but pick younger transgenic F1s as candidate integrants (i.e. see L4 F1s, but single only L3s).
Your marker is undergoing sporadic silencing. This could be happening in the array already but you would not be looking for it. (You could easily test to see if transgenics are segregated from an apparent non-transgenic from a “99%” line.)
Your UV bulbs are old. The bulbs do “drift” over time, even when they are not used. You can try replacing the bulbs with new ones. If I recall, the sensor that measures exposure inside the chamber is a just solar cell that measures visible light, and the machine is inferring UV exposure from that. The bulbs are still good enough to kill though, so you see lethality but no integrants.
Try another irradiation source. Gamma rays (about 3000-3500 rad) are generally more reliable. Typical sources are Cs-137 (most desirable) and Co-60.
Maybe the 99% array is good enough for your needs?
And the things you probably don’t want to do just yet:
Try another transgene marker, e.g. unc-119 rescue, neomycin resistance, etc. A neat way to get integrants without even trying is to generate your strain as an array in an unc-119; him-8 background (marking the array with unc-119 rescue). If you keep chunking such a strain for many generations, it can become spontaneously integrated and become homozygous because of the Him and increased fitness of the integrants.
Try microparticle bombardment or MosSCI.
Use CRISPR/Cas9 to make your transgene at the endogenous locus (obviously depends on the application).
Consider using a co-injection marker that makes worms more healthy and selecting transgenic animals (unc-119, lin-15, etc). You may be picking the brightest animals, but the integrants may be significantly less so and you are selecting against them. By using phenotypic rescue, you may bypass some bias for bright.
Similar to what Morris mentioned in his comment, I recall that Scott Cameron had a related experience integrating a version of lin-11::gfp the old-fashioned, labor-intensive way that involved irradiating and then picking a LOT of F1s to individual plates, and then individual F2s from each F1 plate. He tried very hard and never got an integrated line, until he started picking pretty much every animal and prioritizing the sick, the weird, and the slow-growing. Upon doing so, he found that integrated lines of this transgene had a severe Egl phenotype not readily seen with the extrashromosomal array (presumably having to do with either or both the increased dosage from having two copies of the integrated array, and the lack of mosaicism). Once he knew about this he could repeat the integration prioritizing Egl candidates, and got a lot of integrants.
You might try re-injecting at a lower concentration, and consider whether you might be overlooking integrant homozygotes because they’re unwell.
Following up on what Morris said, I have used an old Stratalinker. Rather than relying on the setting, I just piloted a bunch of doses and went with those that caused some sterility but not a lot. That might be the best way to maximize your dose; you may be just using a weak dose…
If you are using Pmyo-2::mCherry you could be getting toxicity. We have had a couple of integrated transgenes with the myo-2 promoter and GFP or mCherry that were too strong: the pharynges were like little flashlights. And these lines looked like Eat animals: starved and slow growing. So be careful not to select for brighter animals when you pick. Also, mCherry in the pharynx seems to cause more toxicity than GFP, so be careful!
If not using the myo-2 promoter, I doubt it matters.